Olaparib-Loaded Iron Oxide Nanoparticles for the Transgenic Treatment of Triple-Negative Breast Cancer (TNBC): Integrating Targeted Therapy and MRI Imaging: A Review

INTRODUCTION
Triple-negative breast cancer (TNBC) accounts for 10–15% of all breast cancers and is associated with poor prognosis due to its aggressive biology, metastatic potential, and limited treatment options. Unlike hormone receptor-positive or HER2-positive subtypes, TNBC lacks actionable molecular targets, leaving chemotherapy as the mainstay of treatment [1]. However, chemotherapy often fails to eradicate residual disease, leading to relapse of patients within five years. The discovery of synthetic lethality between PARP inhibitors and BRCA mutations has revolutionized the management of BRCA1/2-mutated TNBC, but only 10–20% of TNBC cases harbor these mutations [2]. Furthermore, PARP inhibitors like Olaparib exhibit suboptimal pharmacokinetics, including rapid clearance and dose-limiting hematologic toxicity [3]. Nanoparticle-based drug delivery systems, particularly iron oxide nanoparticles (IONPs), have emerged as a promising solution to enhance drug bioavailability, reduce off-target effects, and integrate diagnostic imaging [4]. 
This article evaluates the convergence of Olaparib and IONP technology, focusing on their synergistic potential to overcome TNBC’s therapeutic and diagnostic challenges.  
To address these challenges, this review highlights the innovative integration of Olaparib with iron oxide nanoparticles (IONPs) as a multifunctional theranostic platform. Unlike conventional delivery systems, Olaparib-loaded IONPs uniquely combine pH-responsive drug release, active tumor targeting (e.g., via folate or EGFR ligands), and MRI-guided monitoring into a single nanoscale construct. This approach not only enhances intratumoral drug accumulation but also enables real-time visualization of drug delivery and therapeutic response—a feature absents in existing PARP inhibitor regimens. Furthermore, the magnetic properties of IONPs allow external field-guided targeting, bypassing stromal barriers and improving penetration into hypoxic tumor cores. By co-encapsulating Olaparib with siRNA or immune modulators, this platform extends therapeutic utility beyond BRCA-mutated TNBC, offering a synergistic strategy to sensitize homologous recombination-proficient tumors.  
This article aims to critically evaluate the design, efficacy, and clinical potential of Olaparib-loaded IONPs as a transformative solution for TNBC. Specifically, it explores (1) the mechanistic basis for combining PARP inhibition with nanomedicine, (2) advances in IONP engineering to optimize drug loading, tumor targeting, and MRI contrast, and (3) preclinical and clinical evidence supporting their theranostic utility. Additionally, it discusses unresolved challenges, including long-term biocompatibility, scalability, and biomarker-driven patient stratification. By synthesizing interdisciplinary insights from oncology, nanotechnology, and imaging sciences, this review underscores the potential of Olaparib-IONP hybrids to redefine precision therapy in TNBC, bridging the gap between targeted treatment and non-invasive diagnostics in one of oncology’s most formidable malignancies.  

BIOLOGY OF TRIPLE-NEGATIVE BREAST CANCER
Triple-negative breast cancer (TNBC) is a highly heterogeneous malignancy characterized by the absence of estrogen receptor (ER), progesterone receptor (PR), and HER2 expression [5]. Molecular subtyping, based on gene expression profiling, identifies six distinct subtypes under the Lehmann classification: basal-like 1 (BL1), basal-like 2 (BL2), immunomodulatory (IM), mesenchymal (M), mesenchymal stem-like (MSL), and luminal androgen receptor (LAR) [6]. BL1, representing approximately 35% of TNBCs, is driven by proliferative signatures linked to cell cycle genes, such as CCNE1 and CDK6, as well as DNA damage response pathways involving ATR and CHEK1 [7]. BL2 tumors exhibit metabolic addiction, characterized by overexpression of growth factor receptors like EGFR and FGFR1 and enzymes critical to glycolysis, including HK2 and LDHA [8]. The IM subtype is marked by elevated levels of CXCL9 and CXCL10 chemokines and enhanced antigen presentation via HLA-DR and CD80 [9]; however, immune evasion is facilitated by activation of the PD-L1/PD-1 axis and infiltration of regulatory T cells and exhausted CD8+ T cells expressing FOXP3 and LAG-3. Mesenchymal and MSL subtypes are associated with epithelial-mesenchymal transition (EMT), featuring the loss of E-cadherin, gain of N-cadherin, and the activity of transcription factors like SNAI1 and TWIST1 [10]. These subtypes promote metastasis and resistance to anthracyclines through matrix remodeling mediated by enzymes such as MMP2 and LOXL2 [11]. LAR tumors are distinct in their reliance on androgen receptor (AR) signaling and frequent PIK3CA mutations, making them sensitive to AR antagonists such as enzalutamide, while exhibiting resistance to standard chemotherapy [12]. At the genomic level, TNBC frequently harbors TP53 mutations, found in approximately 80% of cases. These mutations, often truncating variants like R175H and R248Q, result in loss of p53’s apoptotic function [13]. Alterations in BRCA1 and BRCA2 genes occur in 15–20% of TNBC cases, with germline mutations accounting for 70% and somatic mutations for 30% of these alterations [14]. Additional homologous recombination deficiencies are driven by mutations in PALB2, RAD51C, and ATM, which contribute to genomic instability [15]. Oncogenic alterations include MYC amplifications observed in 30% of cases, associated with chemoresistance, and PTEN deletions seen in 15%, which activate the PI3K/AKT/mTOR pathway [16]. The tumor microenvironment (TME) plays a significant role in TNBC progression and therapeutic resistance. Hypoxia, mediated by HIF-1α, drives the expression of genes such as CAIX and GLUT1 [17]. Cancer-associated fibroblasts (CAFs) enhance interstitial fluid pressure through the deposition of collagen and fibronectin. CAF-derived exosomes transfer microRNAs like miR-21 and miR-155 to tumor cells, silencing tumor suppressors such as PTEN and BRCA1 [18]. Metabolic interactions between tumor cells and CAFs include lactate transfer via MCT4 and MCT1, fueling oxidative phosphorylation, and the production of glutamine to support nucleotide synthesis [19]. Immune suppression is driven by IDO1+ dendritic cells, which catabolize tryptophan into kynurenine, suppressing T cell function [20]. Pembrolizumab shows limited efficacy in PD-L1-positive TNBC due to low tumor mutational burden and TGF-β-mediated exclusion of cytotoxic T cells [21]. Emerging strategies, including inhibitors targeting EMT transcription factors, collagen crosslinking, and metabolic symbiosis, aim to overcome these resistance mechanisms and improve treatment outcomes [22].

OLAPARIB: MECHANISM OF ACTION AND CLINICAL LIMITATIONS
Olaparib, a potent inhibitor of poly(ADP-ribose) polymerase (PARP) enzymes (PARP1/2), exerts its therapeutic effect by disrupting the base excision repair (BER) pathway, a critical mechanism for repairing single-strand DNA breaks (SSBs) [23]. PARP enzymes detect SSBs, recruit repair proteins, and synthesize poly(ADP-ribose) (PAR) chains to signal downstream repair. By binding to PARP’s catalytic domain, Olaparib prevents PAR synthesis and traps PARP-DNA complexes, converting transient SSBs into persistent double-strand breaks (DSBs) [24]. In tumors with BRCA1/2 mutations or homologous recombination deficiency (HRD), the loss of homologous recombination repair (HRR) renders cells unable to resolve these DSBs, leading to synthetic lethality—a collapse of genomic integrity and apoptotic cell death [25]. Beyond BRCA mutations, HRD can arise from epigenetic silencing (e.g., BRCA1 promoter methylation) or defects in HRR-associated genes (e.g., PALB2, RAD51) [26]. Olaparib has improved progression-free survival in BRCA-mutated TNBC, with landmark trials (e.g., OlympiAD) showing a median PFS of 7.0 months vs. 4.2 months for chemotherapy [27]. However, its efficacy is undermined by pharmacokinetic limitations: oral bioavailability ranges from 10% (fasted state) to 60% (with high-fat meals), and rapid hepatic metabolism by cytochrome P450 enzymes (CYP3A4) necessitates frequent dosing. Systemic exposure correlates with dose-dependent hematologic toxicity, including grade 3/4 anemia (40%) and thrombocytopenia (30%), often requiring dose interruptions [28]. Furthermore, acquired resistance emerges via PARP1 overexpression (amplifying DNA repair capacity), upregulation of drug efflux pumps (ABCB1, ABCC1) [29], or restoration of HRR through secondary BRCA1/2 mutations (“reversion mutations”) or stabilization of replication forks via loss of PTIP or PARI [30]. Tumor-targeted delivery systems, such as nanoparticle encapsulation, are critical to circumvent these limitations by enhancing intratumoral drug accumulation, reducing systemic exposure, and overcoming efflux-mediated resistance [31].  

IRON OXIDE NANOPARTICLES (IONPS) IN CANCER THERANOSTICS
Iron oxide nanoparticles (IONPs) are magnetically responsive materials, typically composed of magnetite (Fe₃O₄) or maghemite (γ-Fe₂O₃), with sizes ranging from 10–100 nm to optimize magnetic properties and biodistribution [32]. Their synthesis varies based on precision requirements. Co-precipitation involves alkaline precipitation of Fe²⁺/Fe³⁺ salts, producing hydrophilic IONPs, while thermal decomposition of organometallic precursors like iron oleate in high-boiling solvents creates monodisperse, hydrophobic IONPs that require further ligand exchange for biocompatibility [33]. Microemulsion methods allow controlled size synthesis in confined nanoreactors, but scalability remains limited [34].  
Post-synthesis, functionalization enhances stability and targeting. Polyethylene glycol (PEG) coatings reduce immune clearance and extend circulation times, while targeting ligands like folate or trastuzumab enable receptor-mediated uptake in tumors overexpressing folate receptors or HER2 [35]. Stimuli-responsive polymers, such as poly(N-isopropylacrylamide), provide temperature- or pH-triggered drug release, improving therapeutic precision [36].  The superparamagnetic core of IONPs provides strong T2/T2 MRI contrast, aiding in high-resolution imaging of tumor margins, angiogenesis, and metastases [37]. As drug delivery agents, IONPs exploit the enhanced permeability and retention (EPR) effect for passive tumor accumulation, while external magnetic fields (0.5–1 T) can improve localization [38]. Their theranostic capabilities integrate diagnostic imaging and targeted therapy; MRI monitors real-time nanoparticle biodistribution, while loaded drugs like doxorubicin or paclitaxel are released at tumor sites, reducing systemic side effects [39].  
Recent advancements highlight multifunctional IONPs co-loaded with siRNA for gene silencing and conjugated with fluorophores for combined MRI and near-infrared fluorescence imaging [40]. These developments expand their utility for multimodal imaging and therapy. However, challenges persist, particularly regarding long-term iron metabolism and renal clearance, which require further study to ensure safety in clinical applications [41].  IONPs represent a versatile platform for cancer diagnosis and therapy, combining magnetic responsiveness with targeted delivery. Their dual functionality and potential for multimodal applications position them as critical tools in advancing personalized medicine [42].  

DESIGN AND DEVELOPMENT OF OLAPARIB-LOADED IONPS
Olaparib incorporation into iron oxide nanoparticles (IONPs) requires precise engineering to achieve optimal drug loading, stability, and controlled release. Various strategies have been employed to address these challenges [43, 44]. Encapsulation of Olaparib within biodegradable polymers such as poly (lactic-co-glycolic acid) (PLGA) or chitosan is commonly achieved using techniques like solvent evaporation or nanoprecipitation [45]. These methods yield drug loading efficiencies (DLE), providing a balance between loading capacity and sustained release. Another approach involves electrostatic adsorption of protonated Olaparib onto negatively charged IONP surfaces, such as those coated with citrate [46]. Covalent conjugation offers a more stable alternative, employing pH-sensitive linkers such as hydrazone or cis-aconityl bonds [47]. These linkers hydrolyze selectively in the acidic tumor microenvironment (pH 6.5–6.8), allowing targeted drug release. For example, carboxylated IONPs can be modified with hydrazide groups that form hydrazone bonds with Olaparib’s ketone groups. This strategy achieves payload retention in neutral pH conditions, drug release in acidic environments, demonstrating effective tumor-specific delivery [48].
Targeting ligands further enhance the specificity of Olaparib-loaded IONPs. Functionalizing the nanoparticle surface with ligands like anti-EGFR antibodies, such as cetuximab, facilitates receptor-mediated uptake in TNBC cells, which commonly overexpress EGFR [49]. This modification increases cellular uptake by threefold compared to non-targeted formulations, improving therapeutic efficacy. Stimuli-responsive systems, such as magnetic hyperthermia-triggered release, provide additional control [50]. In these systems, alternating magnetic fields (AMF) in the range of 100–500 kHz generate localized heat (42–45°C), destabilizing the lipid bilayer or polymer matrix encapsulating Olaparib. This technique synergizes with hyperthermia-induced apoptosis and inhibition of heat shock protein 90 (HSP90), sensitizing tumors to PARP inhibition [51]. In MDA-MB-231 xenografts, these hybrid nanoparticles achieved 60% tumor regression compared to 35% with free Olaparib [52]. However, several challenges remain, including optimizing AMF parameters to prevent damage to healthy tissues and ensuring nanoparticle stability under magnetic or thermal stress [53].

TRANSGENIC TREATMENT APPROACHES IN TNBC
The integration of PARP inhibition with nanoparticle-enabled gene editing and immune modulation signifies a transformative approach to triple-negative breast cancer (TNBC) therapy [54]. This strategy addresses both the genetic vulnerabilities and the immunosuppressive tumor microenvironment (TME), paving the way for more effective treatment paradigms [55]. A key application involves the co-delivery of Olaparib, a PARP inhibitor, with small interfering RNA (siRNA) targeting DNA repair genes such as BRCA2, RAD51, and FANCD2 [56]. These combinations enhance synthetic lethality in homologous recombination-deficient (HRD) tumors. Lipid-coated iron oxide nanoparticles (IONPs) co-loaded with Olaparib and BRCA2-targeting siRNA have shown remarkable synergy, inducing the accumulation of double-strand breaks (DSBs) and reducing IC50 values in BRCA1-wildtype TNBC cell lines like MDA-MB-231. This dual-targeted approach maximizes DNA damage while exploiting existing HRD pathways in cancer cells [57].
Beyond genetic editing, IONPs also address the immunosuppressive TME of TNBC. Silencing of cancer-associated fibroblast (CAF)-derived transforming growth factor-beta (TGF-β) through siRNA-loaded IONPs has shown significant therapeutic benefit. TGF-β blockade reduces collagen I deposition by 70%, alleviating the dense stroma that hinders drug penetration [58]. This stromal modulation enhances Olaparib delivery to tumor cores, improving treatment outcomes. Concurrently, IONPs loaded with agents promoting M2-to-M1 macrophage polarization, such as interleukin-12 (IL-12) mRNA or STAT3 inhibitors, further reprogram the TME. This reprogramming increases CD8+ T-cell infiltration by threefold, creating a more immunogenic microenvironment that enhances the efficacy of immune checkpoint inhibitors like PD-1 antibodies [59]. Immunomodulatory strategies extend to the activation of dendritic cells for improved antigen presentation. IONPs co-loaded with Olaparib and toll-like receptor (TLR) agonists, such as resiquimod (TLR7/8 agonist), have been shown to trigger dendritic cell maturation, boosting T-cell priming and expanding tumor-specific immune responses [60].
Challenges remain, particularly in ensuring the intracellular delivery of nucleic acids and avoiding off-target effects with gene-editing tools like CRISPR-Cas9 [61]. Strategies such as proton-sponge polymers (e.g., polyethyleneimine, PEI) or fusogenic peptides are being employed to enhance endosomal escape, while optimized single-guide RNA (sgRNA) designs aim to improve editing specificity [62]. Bioresponsive IONPs represent another promising development, incorporating HIF-1α-responsive promoters to restrict Cas9 expression to hypoxic TMEs, minimizing off-target activity in normal tissues [63]. Integrating PARP inhibitors with advanced nanoparticle delivery systems enables a multipronged attack on TNBC by targeting its genetic vulnerabilities, reversing chemoresistance, and reshaping the TME. This comprehensive approach has the potential to redefine treatment outcomes for this aggressive cancer type, creating a pathway toward more durable and effective therapies [64].

INTEGRATION OF TARGETED THERAPY AND MRI IMAGING
The theranostic potential of Olaparib-loaded iron oxide nanoparticles (IONPs) lies in their capacity to combine precision therapy with advanced diagnostic imaging, enabling simultaneous treatment and real-time monitoring [65]. This dual functionality addresses critical challenges in oncology, providing targeted drug delivery while employing high-resolution magnetic resonance imaging (MRI) to assess therapeutic responses and adjust strategies dynamically [66].
The superparamagnetic properties of the iron oxide core allow for significant T2/T2 relaxation effects, producing negative contrast (signal voids) on MRI scans [67]. These signal changes directly correlate with nanoparticle accumulation within the tumor, offering precise insights into their biodistribution. Quantitative T2 mapping, which measures local magnetic field variations, can detect intratumoral IONP concentrations with sensitivity as low as 0.1 mM Fe [68]. This allows for verification of nanoparticle targeting efficiency, enabling clinicians to refine external magnetic field parameters (typically 0.5–1 T) to enhance retention and distribution within tumors [69]. Dynamic contrast-enhanced (DCE) MRI, which can utilize either gadolinium-based contrast agents or the intrinsic contrast provided by IONPs, adds another layer of information. DCE-MRI evaluates pharmacokinetic parameters such as Ktrans (the volume transfer constant) and ve (extravascular extracellular space volume fraction), which are critical indicators of Olaparib penetration into the tumor and its effect on the stromal microenvironment [70]. Preclinical studies using orthotopic TNBC models have demonstrated Ktrans after IONP-based therapy correlates intratumoral Olaparib levels and a twofold induction of apoptosis, highlighting the effectiveness of combining imaging metrics with therapeutic interventions [71]. For metastatic TNBC, IONPs co-loaded with Olaparib and gadolinium-DOTA chelates offer dual MRI contrast modalities, enhancing the detection of metastatic lesions. These nanoparticles provide T1 (positive) contrast from gadolinium alongside T2 (negative) contrast from the iron oxide core. This dual-modality imaging significantly improves the detection of small metastases, particularly sub-5 mm lesions in the lungs and liver, which are challenging to identify with conventional imaging techniques [72]. Another innovative advancement includes hypoxia-sensitive IONPs coated with nitroimidazole derivatives [73]. These nanoparticles generate measurable T2 signal changes in response to tumor oxygenation levels, enabling targeted activation of alternating magnetic fields (AMFs) in hypoxic regions [74]. This approach has been shown to improve the precision of Olaparib delivery while reducing off-target exposure [75]. MRI-guided therapy using this approach reduces systemic Olaparib concentrations in critical organs, such as bone marrow and intestinal crypts, mitigating associated hematologic and gastrointestinal toxicity [76]. Diffusion-weighted imaging (DWI) further complements the theranostic capabilities of Olaparib-IONPs. By measuring changes in the apparent diffusion coefficient (ADC), DWI can detect early tumor responses to treatment [77]. Preclinical studies in patient-derived xenograft (PDX) models indicate that a 20% increase in ADC within 48 hours of treatment predicts an 80% reduction in tumor volume [78]. Despite these promising applications, clinical translation faces several hurdles. MRI protocols, including variations in magnetic field strength (1.5T vs. 3T) and pulse sequences (spin-echo vs. gradient-echo), can affect contrast quantification and require standardization [79].

PRECLINICAL AND CLINICAL ADVANCEMENTS
Francesco Schettini et al [80], investigated the effects of a 3-week olaparib (PARP inhibitor) treatment prior to standard neoadjuvant chemotherapy in 35 patients with early-stage breast cancer, including 27 with germline BRCA wild-type (gBRCA-wt) triple-negative breast cancer (TNBC) and 8 with gBRCA-mutated (gBRCA-mut) breast cancer (6 TNBC, 2 HR+/HER2-negative). Assessments included clinical, radiometabolic (via PET/CT), immune, and molecular analyses. Key findings showed partial clinical and radiometabolic responses in 40.7% and 50% of gBRCA-wt patients, respectively, indicating olaparib’s potential benefit beyond BRCA-mutated cases. gBRCA-mut tumors exhibited higher tumor-infiltrating lymphocytes (TILs) and PD-L1 positivity. Clinical responders demonstrated immune shifts: reduced T-regs/T-eff ratio, B cells, and NK cells, alongside increased T-helper cells and CD4/CD8 ratio. Ki67 and TILs remained stable, while PD-L1 positivity non-significantly increased post-olaparib. The study concludes that olaparib may benefit early-stage TNBC regardless of BRCA status and suggests integrating TILs, PD-L1, and BRCA status in future trials to optimize treatment strategies involving PARP inhibitors. 

CONCLUSION AND FUTURE PERSPECTIVES
Despite their promise, Olaparib-loaded IONPs face translational hurdles. Long-term IONP biocompatibility remains uncertain, with concerns about iron accumulation in the liver and spleen. Scalability of Good Manufacturing Practice (GMP)-compliant synthesis and regulatory approval for multifunctional nanoparticles are additional barriers. TNBC heterogeneity necessitates biomarker-driven patient stratification, such as BRCA status or HYAL1 expression, to identify likely responders. Future innovations may include dual-drug-loaded IONPs (e.g., Olaparib + paclitaxel) or immune-modulatory designs incorporating anti-PD-L1 antibodies. Artificial intelligence (AI)-driven MRI analysis could personalize dosing schedules based on real-time tumor response. In conclusion, Olaparib-loaded IONPs represent a paradigm shift in TNBC theranostics, merging precision therapy with advanced imaging to address unmet clinical needs. Collaborative efforts among researchers, clinicians, and regulators are essential to translate this technology from bench to bedside, offering hope for one of oncology’s most formidable challenges.  

CONFLICT OF INTEREST
The authors declare that there is no conflict of interests regarding the publication of this manuscript.